Trisomy

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and mantle cell lymphoma usually are distinctly different in regard to clinical presentation, morphology, immunophenotype, and molecular/genetic findings. In spite of this, select cases may show overlapping characteristics and represent a diagnostic challenge. Cyclin D1 immunohistochemical staining is usually envisioned as a definitive method for resolving this differential diagnosis, with positivity supporting a diagnosis of mantle cell lymphoma. We report a case involving a 58-year-old man with a diagnosis of CLL/SLL for several years. A lymph node excision was performed after increased adenopathy was noted in the cervical region. The excised lymph node showed typical morphologic findings of CLL/SLL, including the presence of characteristic proliferation centers. Cyclin D1 staining, using 3 different antibodies, was present in scattered prolymphocytes and paraimmunoblasts, mostly within proliferation centers. Fluorescence in situ hybridization and conventional cytogenetics demonstrated trisomy 12 and an absence of t(11;14) in lymph node tissue. Focal cyclin D1 expression by immunohistochemistry in nodal CLL/SLL is quite unusual and is discussed as a potential diagnostic pitfall.

(Arch Pathol Lab Med. 2005;129:92-95)

Small lymphocytic lymphoma (SLL) is a relatively indolent, small B-cell neoplasm and is the counterpart of the peripheral blood/bone marrow disorder, chronic lymphocytic leukemia (CLL). In the current World Health Organization (WHO) classification, these entities are unified as CLL/SLL and considered to be functionally equivalent in terms of treatment and prognosis.1 The nodal presentation of CLL/SLL has a characteristic morphology of small lymphocytes with a few larger cells (eg, prolymphocytes and paraimmunoblasts) singly or in small aggregates, giving rise to proliferation centers or "pseudofollicles," a classic finding seen in CLL/SLL. Pseudofollicles can be so characteristic as to make an uncomplicated diagnosis of CLL/SLL quite obvious solely on morphologic grounds. Chronic lymphocytic leukemia/small lymphocytic lymphoma has no pathognomonic molecular or cytogenetic event; however, trisomy 12 is seen in approximately 20% of cases and supports a diagnosis of CLL/ SLL.

Mantle cell lymphoma (MCL) is an important counterpart to CLL/SLL. Both entities share some features, including CDS coexpression and a composition of predominantly small B lymphocytes, but MCL has striking differences in clinical, prognostic, molecular, and genetic features. Patients with MCL present with disseminated disease, extranodal manifestations, aggressive course, and poor responses to standard chemotherapy regimens.2 The most common appearance of MCL is characterized by effacement of the lymph node by a population of small lymphocytes with mature chromatin, similar to CLL/SLL. In MCL, the small lymphocytes tend to have more irregular or cleaved nuclear contours. Prolymphocytes or paraimmunoblasts are not seen in conventional MCL, although similar cells may be seen in a variant.1 Mitotic figures are not uncommon, but proliferation centers are not seen. In spite of these differences, the distinction of CLL/SLL from MCL can be challenging.

Mantle cell lymphoma has a characteristic molecular/ cytogenetic event, namely, the t(ll;14)(ql3;q32) leading to juxtaposition of the immunoglobulin heavy-chain (IgH) gene with the BCL1/CCND1 gene. This translocation leads to an overexpression of the cyclin Dl protein, which can be detected by immunohistochemistry. In MCL, the typical staining pattern of cyclin D1 shows diffuse variable nuclear staining among the neoplastic lymphocytes. The finding of cyclin Dl expression by immunohistochemistry in a lymphoid malignancy is quite sensitive and specific for MCL.124 The immunohistochemical demonstration of cyclin D1 should not be seen in CLL/SLL.

We report a case of lymphoma with classic morphologic features of CLL/SLL and focal expression of cyclin Dl in large cells and proliferation centers. Cytogenetic evaluation by conventional techniques and fluorescence in situ hybridization (FISH) demonstrated the presence of trisomy 12 and an absence of t(11;14).

REPORT OF A CASE

A 58-year-old white man presented with right flank pain and prostatitis. Computed tomographic scans indicated hydronephrosis and prostatic enlargement. A transurethral resection of prostate (TURP) was performed, and pathologic examination of the prostatic specimen revealed involvement by a low-grade lymphoma in addition to acute prostatitis and prostatic stromal hyperplasia. Because of poor morphology in the TURP specimen, a needle core biopsy was performed on an enlarged pelvic lymph node with concurrent flow cytometry. A diagnosis of CLL/SLL was rendered on this specimen. At this time, the patient also was noted to have an elevated white blood cell count (27300/µL). Flow cytometry was performed on the peripheral blood, which showed evidence of CLL.

Initially, the patient did not undergo any treatment for CLL/ SLL. After 1 year, the patient reported enlarging lymph nodes with fever, night sweats, and weight loss. Multiagent chemotherapy (cyclophosphamide, vincristine, and prednisone) was undertaken and was followed by resolution of the symptoms and reduction in size of the lymph nodes, but was stopped after 4 cycles owing to development of neuropathy. Two years later, the patient developed rapid enlargement of cervical lymph nodes. A biopsy of a left anterior cervical lymph node was performed.

MATERIALS AND METHODS

Flow Cytometry Methods

Flow cytometry was performed using 3- or 4-color techniques on individual specimens, as noted (Beckman-Coulter, Miami, Fla). Antibodies for CDS, CD19, CD20, CD23, FMC-7, and κ and λ light chains (Beckman-Coulter) were performed in all cases. CD38 was analyzed in only the most recent presentation.

Immunoperoxidase Methods

Immunoperoxidase stains were performed on paraffin-embedded tissues using an automated stainer (DakoCytomation, Carpinteria, Calif) with a standard streptavidin-biotin-peroxidase complex technique. Antigen retrieval was performed using either citrate buffer/steamer or antigen retrieval solution/pressure cooker. The following stains were used: CD3 (PS1; Ventana Medical Systems, Tucson, Ariz), CD20 (L26), and cyclin D1 (DCS-6; DakoCytomation). On the most recent material, the cyclin D1 was repeated using 2 additional antibodies (Cyclin D1, Novocastra, Newcastle upon Tyne, United Kingdom; and P2D11F11, Ventana).

Cytogenetic Methods

Lymph node tissue was processed for conventional cytogenetics using standard techniques. GTG banding was performed using standard methods, and metaphase cells were analyzed and karyotypes produced using the Cytovision system (Applied Imaging, Santa Clara, Calif).

Fluorescence in situ hybridization analysis for trisomy 12 was performed on interphase cells with the CEP 12 centromere probe (Vysis, Downers Grove, Ill). Fluorescence in situ hybridization analysis for a translocation involving CCND1 and IgH was performed on interphase cells with the LSI CCND1/ICH dual-color, dual-fusion translocation probe (Vysis).

RESULTS

The TURP specimen revealed an infiltrate of small, mature-appearing lymphocytes with focal aggregation. Foci of neutrophils were seen in a background of hyperplastic prostatic stroma. Immunohistochemical analyses were positive for CD20, CDS, and CD23, compatible with a diagnosis of CLL/SLL. Cyclin D1 staining was not identified.

The needle core biopsy of an inguinal lymph node showed predominantly small lymphocytes with irregular nuclear contours. Because of suboptimal fixation, no architectural pattern (eg, nodularity or pseudofollicles) or large cells were appreciable. Three-color flow cytometry showed a predominant population of B cells positive for CD19, CD20 (dim), CDS, CD23, and κ light chain (dim). Immunoreactivity for FMC-7 was negative, and CD38 reactivity was not studied. The flow cytometry of peripheral blood showed an identical immunophenotype.

The most recent cervical lymph node biopsy measured 2.0 X 1.4 X 0.5 cm and was uniformly tan and fleshy. Microscopic examination of the lymph node showed complete effacement of the normal architecture by neoplastic lymphocytes. Interspersed were numerous ill-defined, pale nodules (Figure 1). The predominant population consisted of small lymphocytes with round nuclei and condensed mature chromatin. The paler areas had moderate numbers of larger lymphocytes with features of prolymphocytes and paraimmunoblasts (Figure 2).

Results of 4-color flow cytometry of the lymph node revealed the following: approximately 80% of the cells analyzed consisted of lymphocytes with coexpression of CD19, CDS, CD23, CD20 (dim), and κ light chain (dim), and absent expression of FMC-7. CD38 expression was seen on 62% of the cells.

Cyclin D1 staining, using 3 different manufacturers' antibodies, showed the vast majority of the cells to be negative, with scattered positive cells and foci of positivity in areas corresponding to the proliferation centers (Figure 3). Many of the individual positive cells had large round nuclei, with a single prominent central nucleolus (eg, prolymphocytes and paraimmunoblasts; Figure 4).

Because of the unusual staining pattern and the possibility of MCL, FISH studies and conventional cytogenetics were undertaken. In each FISH study, 200 cells were analyzed. The t(11;14) was considered negative, with 96% of the cells lacking the abnormal fusion (Figure 5, b). The remaining cells had the following signals: 1 incomplete cell with 2 fusion signals, 5 cells with a single fusion due to positional overlap in the nucleus, and 2 cells with incomplete signals. Trisomy 12 (CEP 12 probe) was identified in 52.5% of cells analyzed (Figure 5, a). Conventional cytogenetics of G-banded chromosomes demonstrated a clone of cells with trisomy 12 (47,XY,+ 12) and no other abnormalities (Figure 6).

Additional Cases

Because of the unusual nature of the results, we initiated a review of recent cases diagnosed as CLL/SLL that had been assessed for cyclin D1 expression. Fifteen recent cases were reviewed, and no cyclin D1 staining was identified in proliferation centers or isolated lymphocytes. Rarely, individual spindle cells or endothelial cells with positive staining were identified.

COMMENT

Cyclin D1 is a cell-cycle protein that acts as a type of molecular roadblock in the passage of cells from G1 to S phase/ Cyclin-dependent kinases, which phosphorylate substrates to advance the cell cycle, rely on cyclin proteins to ensure cell progression. Because of its regulatory function of cellular proliferation, cyclin D1 expression by immunohistochemistry can be seen in a variety of normal human tissues and neoplasms.6

Cyclin D1 expression can be seen in myeloma and hairy cell leukemia, with and without the presence of the t(11; 14).78 In addition, cyclin D1 is found at low levels on a variety of B-cell malignancies, including CLL/SLL, when sensitive methods such as flow cytometry or reverse transcription-polymerase chain reaction are used.910 In spite of these findings, in hematopoietic malignancy the detection of cyclin Dl expression by immunohistochemistry has become a cornerstone in the diagnosis of MCL, and its diagnostic specificity and sensitivity are quite high.

Immunophenotype by flow cytometry is considered a useful and practical approach to distinguishing MCL and CLL/SLL; however, even markers with the highest discriminatory value, such as CD5, CD23, and FMC-7, may yield aberrant results in a minority of cases.11,12 The literature contains references to cases positive for t(11;14) with cyclin D1 overexpression that were diagnosed as "atypical CLL." 13,14 In retrospect, it is probable that some of these cases represented MCL with a prominent leukemic component. Still, it is possible that there may be other lymphoid proliferations that share features of both CLL/SLL and MCL, making unusual cases a diagnostic challenge.15

The current case illustrates cyclin D1 overexpression in prolymphocytes and paraimmunoblasts by immunohistochemistry in a lymphoma with clear morphologic features of CLL/SLL and absence of the t(11;14). We speculate that overexpression of cyclin D1 by immunohistochemistry in this case was independent of the t(ll;14) seen in MCL and instead represented constitutive overexpression in a subset of malignant lymphoid cells. Another possibility is that this case of CLL/SLL was in the process of "transforming" to MCL, and that the t(ll;14) was present at levels below detection by the FISH studies.

The current WHO classification of hematopoietic neoplasms stresses the concept of combining clinical, morphologic, immunophenotypic, and molecular/cytogenetic results in arriving at a diagnosis. It is not clearly stated which feature or features are transcendent above the others under different diagnostic circumstances. In many cases, morphology can be considered the gold standard, but this is not universally true. Each case requires careful consideration of all results, especially those that are discordant or unexpected.

There is no doubt that this case is unusual. Although rare, it is possible that this aberrant expression of cyclin D1 in CLL/SLL could represent a source of diagnostic confusion. It is important that pathologists be aware of this potential pitfall in the diagnosis of CLL/SLL and use additional information, such as immunophenotype, cytogenetics, and molecular findings, to resolve difficult cases.

The authors express their thanks to Sherrie Perkins, MD, PhD, and Peter Banks, MD, for their help and suggestions with the staining and manuscript. The authors also thank Lee Ann Baldridge, HT(ASCP)QIHC, for her excellent technical assistance.

References

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7. de Boer CJ, Kluin-Nelemans JC, Dreef E, et al. Involvement of the CCND1 gene in hairy cell leukemia. Ann Oncol. 1996;7:251-256.

8. Troussard X, Avet-Loiseau H, Macro M, et al. Cyclin D1 expression in patients with multiple myeloma. Hematol I. 2000;1:181-185.

9. Elnenaei MO, ladayel DM, Matutes E, et al. Cyclin D1 by flow cytometry as a useful tool in the diagnosis of B-cell malignancies. Leuk Res. 2001;25:115123.

10. Specht K, Kremer M, Muller U, et al. Identification of cyclin Dl mRNA overexpression in B-cell neoplasias by real-time reverse transcription-PCR of microdissected paraffin sections. Clin Cancer Res. 2002;8:2902-2911.

11. Delgado J, Matutes E, Morilla AM, et al. Diagnostic significance of CD20 and FMC7 expression in B-cell disorders, Am / CHn Pathol. 2003:120:754-759.

12. Wohlschlaeger Ch, Lange K, Merz H, Feller AC. Aberrant immunophenotypes of mantle cell lymphomas. Leuk Lymphoma. 2003;44:269-273.

13. Bosch F, lares P, Campo E, et al. PRAD-1/cyclin Dl gene overexpression in chronic lymphoproliferative disorders: a highly specific marker of mantle cell lymphoma. Blood. 1994:84:2726-2732.

14. Cuneo A, Balboni M, Piva N, et al. Atypical chronic lymphocytic leukaemia with tU1;14)(q13;q32): karyotype evolution and prolymphocytic transformation. Br I Haematol. 1995:90:409-416.

15. Matutes E, Carrara P, Coignet L, et al. FISH analysis for BCL-1 rearrangements and trisomy 12 helps the diagnosis of atypical B cell leukaemias. Leukemia. 1999;13:1721-1726.

Dennis P. O'Malley, MD; Gail H. Vance, MD; Attilio Orazi, MD, FRCP

Accepted for publication August 26, 2004.

From the Departments of Pathology and Laboratory Medicine, Division of Hematopathology (Drs O'Malley and Orazi) and Medical and Molecular Genetics (Dr Vance), Indiana University School of Medicine, Indianapolis.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Dennis P. O'Malley, MD, Indiana University School of Medicine, Department of Pathology and Laboratory Medicine, 702 Barnhill Dr, Riley 0969, Indianapolis, IN 46202 (e-mail: dpomalle@ iupui.edu).

Copyright College of American Pathologists Jan 2005
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